Vaccines represent one of the seminal developments in our ongoing battle against disease. Vaccination is still the best defense against existing, novel, and manipulated pathogens. The earliest whole-cell vaccines were prepared by inactivating a given pathogen using chemical or heat processes. Whole-cell vaccines have significant advantages over attenuated and subunit vaccines. Chemical or thermal inactivation of the pathogen is simple and inexpensive, and provides rapid access to a vaccine. Both subunit vaccines and attenuated vaccines require considerable time and expense before they can be put to use. Despite the advantages of chemical inactivation, chemically inactivated vaccines sometimes fail to elicit robust and protective immune responses [2-4]. The addition of adjuvants to these preparations may boost the immune response, but immunity is still insufficient in many cases and may require frequent boosting.
Many complications associated with chemically inactivated vaccines arise from the simple fact that inactivation alters the chemical properties of key antigens required to elicit a protective immune response. The development of a rapid, inexpensive, and effective process to inactivate a pathogen while maintaining the integrity of its antigens would represent a powerful new tool in vaccine development.
Recent work has effectively demonstrated that microbes inactivated by a non-denaturing process do, in fact, elicit more robust immune responses than chemically inactivated pathogens [5]. “Ghosts” as they are known colloquially, are the empty shells of microbes that have been inactivated by the controlled expression of the PhiX174 lysis gene “E” [6]. Essentially the cytoplasmic contents of the cells are expelled via the transmembrane tunnel formed by the lysis protein [6]. Vaccines prepared through this genetic manipulation have been shown to be superior to chemically inactivated pathogens, most likely due to the non-denaturing inactivation procedure [3]. Moreover, it is hypothesized that the more robust immune response is not simply a function of individual proteins, but also is related to the route of antigen presentation.
Cell walls remain largely intact, native surface antigens are preserved, and bioadhesive properties are likely maintained in ghost vaccine preparations. All of these characteristics endow ghost vaccines with inherent adjuvant properties that contribute to protective immune responses [3, 7-17]. The usefulness of the bacterial ghost system is extended by inactivating bacteria expressing antigens that are derived from other pathogens. The end result is a vaccine with inherent adjuvant properties that is protective against any number of desired bacterial, viral, protozoan, and fungal pathogens [12, 13, 15, 16, 18]. There are concerns about the endotoxicity of lipid A and lipopolysaccharide (LPS) in these whole cell vaccines. However, it has been demonstrated that endotoxicity is not a real limit to the use of ghost vaccines [17].
Despite its promise, the ghost vaccine technology exhibits a number of drawbacks. The first of these concerns centers on safety. The phage lytic system employed typically results in only a 4-log reduction in colony forming units (CFU) [5]. The remaining organisms must be inactivated by further processing. This may or may not be the case. The ghost system uses an additional kill mechanism to inactivate the remaining survivors [5]. This layering of genetic systems in the ghost technology is a cause for additional concern. Because these genetic systems are maintained within the chosen cells by selection on various antibiotic containing media [19, 20], lateral transfer of antibiotic resistance to other pathogens within an individual is a possibility [21].
In addition to safety concerns, the ghost system only works with Gram-negative bacteria. Furthermore, genetic manipulation of additional serotypes may be required to generate a broadly protective vaccine. Therefore, the applicability of the ghost technology is limited to the gram-negative bacteria that are tractable to genetic manipulation. These limitations preclude a significant number of pathogens, notably: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus pyrogenes, Enterococcus spp., Bacillus anthracis, Bacillus cereus, Lactobacillus spp., Listeria monocytogenes, Nocardia spp., Rhodococcus equi, Erysipelothrix rhusiopathiae, Corynebacterium diptheriae, Propionibacterium acnes, Actinomyces spp., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, and Peptostreptococcus spp. The applicability of ghost vaccine technologies is further limited by its failure to inactivate spores, which are insensitive to induction of lysis genes due to their dormant nature.
Whole-cell vaccines produced on the ghost vaccine technology are superior to chemically inactivated pathogens, but cannot be developed rapidly. Even if the microbe is previously known, considerable time and expense are required to generate a new ghost vaccine for a given pathogen, especially for novel or genetically intractable pathogens. The present invention does not require introduction of a phage lysis gene and induction of a lytic program.
The need for new and broadly applicable inactivation technologies is exacerbated by the very nature of biological weapons. The pathogens that are, or may be employed as bio-warfare and bio-terror agents such as Anthrax, Tularemia, Botulism, Plague, Epsilon toxin, Q fever, enterotoxin B, Typhus fever, Melioidosis, and Brucellosis are not usually endemic diseases in humans. As such there is very little, if any, commercial advantage to generating vaccines using expensive and time-consuming techniques. An appealing alternative that would speed the production of such vaccines and enable quick response to emerging serotypes is an inactivation technology that in and of itself generates high quality vaccines. Bacterial inactivation by supercritical CO2 represents such a technology. The technology for using supercritical CO2 is well-known and has been adapted to large industrial applications, including the extraction of natural compounds from plant materials [22] and detoxification of contaminated soil [23]. Supercritical CO2 applications have also found their way into medical circles as a process for bone de-lipidation [24], drug manufacture [25], and sterilization among others [1]. The first attempts to use supercritical CO2 as a sterilant resulted in inadequate levels of inactivation [26].
Recently, in U.S. Pat. No. 6,149,864 to Dillow et al. (the entire content of which is expressly incorporated hereinto by reference), the use of supercritical CO2 was disclosed as an alternative to existing technologies for sterilizing a wide range of products for the healthcare industry with little or no adverse effects on the material treated. Specifically, the Dillow '864 patent disclosed the inactivation of a wide range of vegetative microbial cells using supercritical carbon dioxide with agitation and pressure cycling. However, only one spore-forming bacterium was investigated in the Dillow '864 patent, specifically, B. cereus. No disclosure appears in Dillow '864 patent regarding the efficacy of the therein suggested techniques using currently accepted bio-indicator standards used to judge sterilization (i.e., B. stearothermophilus and B. subtilis). Subsequently, however, other investigators achieved only a 3.5-log reduction in B. subtilis spore forms using the process disclosed in the Dillow '864 patent [27].
In addition to bacterial inactivation, viral inactivation is realized using supercritical CO2 [28]. Moreover it has been shown that sterilization by supercritical CO2 does not affect the properties of a biodegradable polymer (PLGA) and leaves bacterial cells intact [1].
It would therefore be desirable if processes could be provided whereby organisms are inactivated utilizing near or supercritical CO2 for the purpose of generating whole-cell therapeutic agents. It is towards fulfilling such a need that the present invention is directed.